Vasopressin (VP) and oxytocin (OT) secretion from the posterior pituitary occurs as a result of a variety of neurotransmitters and neuropeptide afferents to the supraoptic and paraventricular nuclei in the hypothalamus. Many of these afferents co-localize and co-release multiple neuroactive substance. In evaluating the responses to agents colocalized in the A1 catecholamine neurons (e.g. norepinephrine, ATP, neuropeptide Y (NPY), and substance P (SP), we found evidence for differential potentiation of hormone release by these substances as well as a potent and sustained response to SP. Specifically, combined exposure to SP and ATP or to NPY and phenylephrine (PE, an alpha adrenergic receptor agonist) resulted in synergistic stimulation of VP and OT release. The goals of the current proposal are to determine the relative importance of NPY and SP in eliciting VP and OT responses to physiological challenges and to elucidate the cellular mechanisms responsible for these synergistic responses. Since the A1 pathway transmits information about moderate decreases in blood pressure/volume to the VP and OT neurons, SP and NPY may be important to this physiological response. Aim 1 will test the hypothesis that release of SP and NPY from A1 terminals is required for activation of VP neurons in response to hypotension. The effect of unilateral supraoptic nucleus (SON) injections of SP and NPY receptor antagonists on Fos expression in SON will be assessed following hemorrhage or an acute osmotic stimulus. Aim 2 will evaluate the cellular mechanisms responsible for the synergistic responses to co-exposure to SP/ATP and NPY/PE. Explants of the hypothalamo-neurohypophyseal system will be used to: identify the receptor types required for synergism; determine the roles of protein kinase C and IP3-mediated Ca++ release; and evaluate the role of gene transcription. Aim 3 will test the hypothesis that SP and NPY receptor expression in SON is altered by stimulation of the A1 pathway. Western blots, immunocytochemistry, and in situ hybridization will be used to assess the expression of the neurokinin 3 tachykinin receptor and Y1/Y5 NPY receptors in rats following water deprivation, chronic saline ingestion, and hemorrhage. Aim 4 will test the hypothesis that dehydration-induced increases in Y1/Y5 receptor expression in SON alter the VP and OT response to NPY. The VP, and OT response to NPY will be evaluated using HNS explants obtained from rats exposed to chronic water deprivation. Inadequate or inappropriate VP secretion contributes to pathologies such as orthostatic hypotension and congestive heart failure. Understanding the role of specific neurotransmitters in relaying hemodynamic information to the VP and OT neurons is central to manipulating the system for treatment of pathological conditions.